Role And Mechanism Of M2 Macrophages In Tendon-bone Healing

The tendon-bone junction is a critical site for force conduction from muscle to bone and is also a vital structure providing a transition from soft to hard tissue.Owing to stress concentration,this site is susceptible to rupture,resulting in a high clinical incidence and disability rate.The normal tendon-bone junction constitutes the typical four-layer structure containing the bone tissue,calcified fibrocartilage layer,non-calcified fibrocartilage,and tendon tissue.However,following an injury to this site,massive scar tissue accumulates at the tendon-bone interface,resulting in its poor biomechanical properties.Consequently,the re-rupture rate after tendon-bone reconstruction is as high as 20-94%.Previous studies have shown that the fibrocartilage layer is a crucial biological structure and its regeneration is associated with healing quality after tendon-bone injury.Therefore,strategies to promote fibrocartilage formation during tendon-bone healing has become a research hotspot.Similar to that in other tissues,tendon-bone healing is a sequential process consisting of inflammatory,proliferative,and remodeling phases.During this process,multiple types of immune cells are known to be involved,including neutrophils,macrophages,monocytes,and mast cells.Studies have demonstrated that macrophages play a crucial role in tissue repair and regeneration.At the early stage of tissue healing,macrophages are mainly of the pro-inflammatory(M1)phenotype,secreting pro-inflammatory factors such as IL-1?,TNF-?,and IL-6 and functioning to defend against pathogens.Afterward,macrophages polarize towards the anti-inflammatory(M2)phenotype and mainly secrete IL-10,arginase-1(Arg1),and TGF-? to promote tissue repair.As suggested in a previous study,the growth factors secreted by M2 macrophages promote cartilage repair at the joints and boost the chondrogenic differentiation of mesenchymal stem cells(MSCs).In addition,macrophage depletion is disadvantageous to tendon-bone healing.However,the role and mechanisms of M2 macrophages in tendon-bone healing remain unclear.In this study,we aimed to investigate the functional role of macrophages in the process of tendon-bone healing.We found that macrophage depletion resulted in poor tendon-bone healing.However,macrophages polarization towards the M2 phenotype promoted tendon-bone healing.We discovered that M2 macrophages secrete TGF-?3 to promote fibrocartilage formation,thereby accelerating tendon-bone healing.In addition,our findings may provide a potential new therapeutic strategy for tendon-bone healing in the future.Objectives1.To determine the relevance of macrophages change and the fibrocartilage formation in tendon-bone healing.2.To elucidate the effect of M2 macrophages on tendon-bone healing and the fibrocartilage formation.3.To explore the mechanism of M2 macrophages promoting fibrocartilage formation in tendon-bone healing.4.To study the effect of BMSCs exosomes on macrophage polarization and fibrocartilage formation during tendon-bone healing.Methods1.To determine the relevance of macrophages change and the formation of fibrocartilage in tendon-bone healing.We established the tendon-bone healing model,and analyzed the phenotypic changes of macrophages at different time points by immunofluorescence staining and PCR analysis.Investigating the fibrocartilage formation at different time points by immunofluorescence staining and PCR analysis.To examine the relationship between phenotypic transformation of macrophages and fibrocartilage formation during tendon-bone healing,we depleted macrophages during the tendon-bone healing process.We observed the effect on the fibrocartilage formation at the interface of tendon-bone,and analyzed the effect of lack of macrophages on biomechanics after healing.2.To elucidate the effect of M2 macrophages on tendon-bone healing and the fibrocartilage formation.First of all,we established a mouse tendon-bone healing model,and induced macrophages to polarize toward M2 type by local injection of IL-4/13.Histology and immunofluorescence staining were used to evaluate the changes of fibrocartilage at the interface of tendon-bone.The biomechanical evaluation was used to assess the effect of M2 polarized macrophages on tendon-bone healing.3.To explore the mechanism of M2 macrophages promoting fibrocartilage formation in tendon-bone healing.First of all,we sorted M1 and M2 macrophages during tendon-bone healing by flow cytometry,screened the cytokines that regulate the chondrogenic differentiation of stem cells by PCR and ELISA analysis.Next we verified that M2 macrophages promoted fibrocartilage formation at the tendon-bone interface by secreting TGF-?3.4.To study the effect of BMSCs exosomes on macrophage polarization and fibrocartilage formation during tendon-bone healing.The exosomes was isolated from BMSCs and injected locally into the tendon-bone healing site.The effect of BMSC exosomes on macrophages polarization and fibrocartilage formation in tendon-bone healing were observed.Results1.The formation of fibrocartilage during tendon-bone healing are related to the changes of macrophages.It was found that there were mainly M1 macrophages in the interface tissue on the 3 day after the operation and M2 macrophages on the 7 day after the operation.The expression of factors promoting chondrogenic differentiation increased on the 7 and 10 day after the operation,and fibrocartilage formation began at the 14 day after the operation.At 1 month after model establishment,HE staining showed that the macrophage depletion group has less chondroid-shaped cells and collagen deposition at the tendon-bone junction.In the macrophage depletion group,the Safranin O-positive area was significantly reduced compared with that in the control group.These results show that the absence of macrophage was detrimental to fibrocartilage formation.Biomechanical testing indicated that the maximum force,stiffness,stress at failure,and modulus decreased after macrophage depletion.The results showed that there was a correlation between macrophages and fibrocartilage formation in the process of tendon-bone healing.2.M2 macrophage promotes fibrocartilage formation and enhances the biomechanical properties in the tendon-bone healing.At 1 month after surgery,Safranin O-Fast Green staining showed that the cartilage tissue abundance was markedly increased by M2 macrophage polarization.The expression of collagen II at the tendon-bone junction was increased in the M2 macrophage polarization group compared with the control group.In addition,biomechanical testing demonstrated that the maximum force,stiffness and stress at failure were notably increased by M2 macrophage polarization.3.M2 macrophages promote fibrocartilage formation by secreting TGF-?3 during tendon-bone healing.The expression of TGF-?1,TGF-?3,IGF-1 and IGF-2 was markedly increased by M2 macrophage polarization induction.The ELISA analysis results showed that M2 macrophages secreted more TGF-?1 and TGF-?3 than non-M2 macrophages.At 1 month after model establishment,Safranin O-Fast Green staining demonstrated cartilage tissue at the tendon-bone junction markedly increased in administered recombinant TGF-?3 group.However,chondrogenesis was prevented by the local administration of the recombinant TGF-?3 neutralizing antibody.The expression of collagen II and aggrecan at the tendon-bone junctions was increased by the administration of TGF-?3;however,the changes in collagen II and aggrecan expression were reversed by TGF-?3 neutralizing antibody injection.4.BMSCs exosomes induces macrophage polarization to M2 type and promotes fibrocartilage formation during tendon-bone healing.In the early phase,we observed significantly higher numbers of M2 macrophages and more anti-inflammatory and chondrogenic-related factors in the hydrogel+BMSC-Exos group compared with the control group and the hydrogel group.The M1 macrophages and related proinflammatory factors decreased.Cell apoptosis decreased in the hydrogel+BMSC-Exos group,while cell proliferation increased;in particular,the CD146+ stem cells substantially increased.At 1 month after surgery,there was more fibrocartilage in the hydrogel+BMSC-Exos group than the other groups.Biomechanical testing showed that the maximum force,strength and elastic modulus were significantly improved in the hydrogel+BMSC-Exos group.ConclusionThis paper comprehensively expounds on the role and mechanism of macrophages in tendon-bone healing.Firstly,we found that there is a correlation between macrophages and fibrocartilage formation in tendon-bone healing.The macrophage depletion in tendon-bone healing is not conducive to fibrocartilage formation and reduces biomechanical properties.Later it has been found that macrophages polarization toward M2 type promote fibrocartilage formation in tendon-bone healing.In addition,the screening and verification the cytokines related to chondrogenic differentiation in tendon-bone healing show that M2 macrophages promote fibrocartilage formation by secreting TGF-?3.Finally,it was found that BMSCs exosomes could regulate the polarization of macrophages to M2 type during tendon-bone healing,so as to promote the formation of fibrocartilage and improve the biomechanical properties.SignificanceIn this study,we elucidated the role of macrophages in tendon-bone healing and the mechanism of M2 macrophages promoting fibrocartilage formation.The conclusion of our study provides a scientific basis for promoting tendon-bone healing and a therapeutic strategy for improving the intensity of tendon-bone healing in the clinic.